Method for multifragment in vivo cloning and mutation mapping

ABSTRACT

The subject invention relates to a method referred to as multifragment in vivo cloning (MFIVC). In the method, the polymerase chain reaction or the cleavage by restriction enzyme(s) are used to generate a series of double-stranded DNA fragments. Each fragment contains a region homologous to a portion of the fragment to which it is to be joined. These homologous regions undergo recombination in vivo following transformation into a host with efficient and precise homologous recombination (such as the yeast  S. cerevisiae ). A series is designed so that the last fragment in the series contains a region homologous to a portion of the first fragment in the series, thus forming a circular DNA molecule after recombination in vivo. A circular DNA molecule can be selected in vivo if the circular DNA molecule created contains both a suitable DNA replication origin and a suitable marker for genetic selection. A series may be designed so that the first and last fragment in the series contain telomeric sequence elements, forming a linear DNA molecule with telomeric sequence elements at its ends, after recombination in vivo. One preferred embodiment of this method includes a means for mapping a phenotypically expressed mutation within a gene. A second embodiment of this method includes a means for constructing plasmids using DNA cassettes. A third embodiment of this method includes a means of reasserting mutations in a double-stranded DNA molecule. The invention also includes kits containing reagents for conducting the method.

This application is a Division of Ser. No. 08/584,322 filed Jan. 13,1996 and now U.S. Pat. No. 5,976,846.

BACKGROUND—FIELD OF INVENTION

The present invention is in the field of recombinant DNA technology.This invention relates to a process for assembling multiple DNAfragments in vivo, and to the molecules employed and produced throughthis process. Thus, the method can be used for the rapid generation ofrecombinant constructs and for mapping phenotypically expressedmutations.

BACKGROUND—DESCRIPTION OF PRIOR ART

Two of the fundamental tools of the field of recombinant DNA technologyare the ability to recombine DNA, and the ability to localize (or map)the position of phenotypically expressed mutations.

Methods to assemble DNA fragments into plasmids that can replicate invivo are of fundamental importance in the field of recombinant DNAtechnology. Such methods can be used, for example, to construct aplasmid bearing a particular gene being studied. Typically, suchmethodologies involve the introduction of a nucleic acid fragment into aDNA or RNA vector, the clonal amplification of the vector (and therecovery of the amplified fragment). Examples of such methodologies areprovided by Cohen et al. (U.S. Pat. No. 4,237,224), and Maniatis, T. etal., Molecular Cloning (A Laboratory Manual), Cold Spring HarborLaboratory, 1982.

The desire to increase the utility and applicability of such methods isoften frustrated by the lack of (suitable) restriction enzyme sitespresent at desired locations and, even when suitable restriction sitesare present, by the methodological complexities involved in complex3-way and 4-way ligations (such as sequential digestions, fragmentisolation, and buffer incompatibility). Hence, it would be highlydesirable to develop a general, simple, and rapid method to assemblemultiple DNA fragments.

The polymerase chain reaction (PCR) technique was conceived anddeveloped by the Cetus Corporation to provide for specific amplificationof discrete fragments of DNA in order to allow simplified detection andpurification of nucleic acid fragments initially present in a particularsample in only picogram quantities (Salki, et al. Science 230:1350-1354,1985). The basic method is based on the repetition of three steps, allconducted in a successive fashion under controlled temperatureconditions: (1) denaturing the double-stranded template DNA; (2)annealing the single-stranded primers to the complementarysingle-stranded regions on the template DNA; and, (3) synthesizingadditional DNA along the templates by extension of the primer DNAs withDNA polymerase after 4 to 25 cycles of these steps; as much as a100,000-fold increase in the amount of the original DNA is observed(Oste, BioTechniques 6:162-167, 1988). Reviews of the polymerase chainreaction are provided by Mullis, K. B. Cold Spring Harbor Symp. Quant.Biol. 51:263-273 (1986); and Mullis, K. B. et al. Meth. Enzymol.155:335-350 (1987), which are incorporated herein by reference.

More recently, the PCR technology has been used for mutagenesis ofspecific DNA sequences and for other directed manipulations of DNA. Forinstance, PCR technology has been used to engineer hybrid (chimeric)genes without the need to use restriction enzymes in order to segmentthe gene prior to hybrid formation. In this approach, fragments that areto form the hybrid are generated in separate polymerase chain reactions.The primers used in these separate reactions are designed so that theends of the different products of the separate reactions containcomplementary sequences. When these separately produced PCR products aremixed, denatured, and reannealed, the strands having matching sequencesat their 3′-ends overlap and act as primers for each other. Extension ofthis overlap by DNA polymerase produces a molecule in which the originalsequences are spliced together to form a hybrid gene. Thus, this methodrequires four primers to construct a deleted, hybrid DNA molecule.Likewise, the method requires six primers and three rounds of PCR inorder to construct a chimeric molecule (Horton, et al., Gene 77:61-68,1989).

Recently Jones (U.S. Pat. No. 5,286,632) has described a method that canbe used to join two DNA molecules. In this method, the polymerase chainreaction is utilized to add double-stranded regions to both ends of aninsert DNA homologous to the ends of a linear vector. These homologousends undergo recombination with a linear vector in vivo followingtransformation of Escherichia coli. This method can be used to introducemutations into a preexisting vector but can not be used to rearrange(recombine) preexisting mutations present on separate DNA inserts. Thismethod can be used to join at most two DNA molecules:, this is asignificant disadvantage since it is often desirable to join 3 DNAmolecules. This method also requires that the homologous regions whichundergo recombination be located at the ends of the DNA moleculesamplified by PCR, thus necessitating the design and synthesis of primersunique for each particular pair of gene and cloning vector. Anadditional drawback of this method is a requirement to either purifyboth PCR fragments after amplification or to cut the template plasmidswith a restriction enzyme that recognizes a site outside of the regionto be amplified prior to amplification. An additional disadvantage ofthe in vivo cloning procedure described in the Jones patent (U.S. Pat.No. 5,286,632) is the extreme inefficiency of the recombination events;the recombination is less than one in 10,000,000-fold as efficient astransformation of intact plasmid.

Muhlrad et al. have described a method to introduce mutations into genescloned into plasmids that replicate in yeast. (Muhlrad, D., Hunter, R.,and Parker, R. Yeast 8:79-82, 1992). This method can not be used torearrange (recombine) preexisting mutations present on DNA fragments.This method can be used to join at most 2 DNA molecules; this is asignificant disadvantage.

Oliner et al. have described another method of in vivo cloning utilizingan E. coli strain with enhanced in vivo recombination (Oliner, J. D.,Kinzler, K. W., and Vogelstein, B. Nucleic Acids Research 21:5192-5197,1993). Although this method has an improved transformation efficiencycompared to the methods described in the Jones patent (U.S. Pat. No.5,286,632), the transformation efficiency is still approximately100,000-fold lower than that of intact plasmid DNA transformed into E.coli, and the likelihood of efficient trimolecular and higher orderrecombinations would be extremely low.

Willem P. C. Stemmer (Stemmer, W. (1994) Nature 370, 389-391) hasdescribed a method for in vitro homologous recombination called DNAshuffling. In this method, pools of selected mutant genes are recombinedin vitro by random fragmentation and PCR reassembly. In this method, therecombined molecules had to be cloned into an appropriate vector beforetransformation into E. coli and subsequent selection and analysis. Thisrecloning step had to be repeated for each iteration of the selectionprocess.

An alternative method for recombinational mapping of plasmid-borne genesin yeast has been described (Kunes, S., Ma, H., Overbye, K., Fox, M. S.,& Botstein, D. (1987) Genetics 115: 73-81). The method of Kunes et al.relies upon the incidence of loss of a plasmid-borne mutation toidentify its location. This method is based on a statistical analysisand provides a genetic map distance of a mutation from the end of a DNAfragment, the position of the end being determined by the fortuitouslocation of a restriction enzyme recognition site. This method is basedon the loss of a plasmid-borne mutation to identify its location; in theprocess the mutation is lost rather than subcloned.

Ma et al. have presented a method for constructing plasmids in yeast byhomologous recombination (Ma, H., Kunes, S. Schatz, P. J. and Botstein,D., Gene 58: 201-216). However, this method can not be used to rearrange(recombine) preexisting mutations present on DNA fragments. This methodcan be used to join at most 2 DNA molecules; this is a significantdisadvantage.

Degryse et al. in vivo cloning by homologous recombination in yeastusing a two-plasmid-based system Yeast 11,629-640. This method can beused to join at most 2 DNA molecules; this is a significantdisadvantage. An additional drawback of this method is the requirementthat one must first construct, using conventional methods, the twoplasmids used in the two-plasmid-based system.

OBJECTS AND ADVANTAGES

We describe a method to join multiple DNA fragments. This methodutilizes recombination between sequence elements present at the end(s)of a series of DNA fragments to specify the junctions and usesrecombination enzymes present in vivo to accomplish the joining. Thismethod is superior to previous methods because it does not require theuse of any specific DNA restriction or modification enzymes in vitro,other than those used in the PCR process.

This method is superior to previous methods because it can be used tojoin 3 or more DNA fragments precisely in a single procedure. This is anessential step of a process we describe in detail termed cassette-basedcloning, that can be used to construct plasmids rapidly from premadeparts that can be combined in a multitude of combinations.

This method is superior to previous methods because it can be used torecombine mutations present on separate DNA inserts into a single newinsert, carried on a cloning vector. This is an essential step of aprocess we describe in detail termed directed-evolution, that can beused to incrementally modify the function (activity, affinity, thermalstability or some related property) of a protein (or catalytic RNA orDNA).

This method is superior to previous methods because it can be used tophenotypically map mutations to a particular physical DNA fragment, andin the same process subclone the phenotypic mutation away from any othermutations that might be present.

This method is superior to previous methods because it can be used toconstruct vectors that stably transform yeast and that contain onlyendogenous yeast sequences. Plasmids constructed in yeast and lackingany foreign (non-yeast) DNA might prove desirable in some circumstanceswhere more traditional, foreign DNA-containing plasmids would beunacceptable because of regulatory prohibitions and/or social stigmasassociated with the use of recombinant DNA.

Other objects and advantages will become apparent from thespecifications and drawings.

DRAWING FIGURES

FIG. 1: A multifragment cloning method based on homology introduced atone end of each fragment in a series.

FIG. 2: A multifragment cloning method based on homology introduced atboth ends of each fragment in a series.

FIG. 3: A method of recombinational mapping of mutations.

FIG. 4: Recombinational mapping of mutations, a specific example.

FIG. 5: Cassette-based cloning methods.

FIG. 6: Directed-evolution based on multifragment cloning.

REFERENCE NUMERALS IN DRAWINGS

FIG. 1. A multifragment cloning method based on homology introduced atone end of each fragment in a series.

10 A primer homologous to one end of the top strand of a yeast markergene (11).

11 A DNA fragment that is a source of a yeast marker gene.

12 A primer with a 3′ end complementary to the end of the top strand ofthe yeast marker gene (11), distal to the primer (10), and with a 5′ endcomplementary to a primer (13).

13 A primer homologous to one end of the top strand of a yeastreplication sequence element (14).

14 A DNA fragment that is a source of a yeast replication sequenceelement.

15 A primer with a 3′ end complementary to the end of the top strand ofthe yeast replication sequence element (14), distal to the primer (13),and with a 5′ end complementary to a primer (16).

16 A primer homologous to one end of the top strand of a gene to becloned (17).

17 A DNA fragment that is a source of a gene to be cloned.

18 A primer with a 3′ end complementary to the end of the top strand ofthe gene to be cloned (17), distal to the primer (16), and with a 5′ endcomplementary to the primer (10).

20 A DNA fragment [generated using the PCR process] that bears the yeastselectable marker and an end homologous to a fragment (22) that bearsthe yeast replication sequence element, introduced as a result of thePCR process using the primer (12).

22 A DNA fragment [generated using the PCR process] that bears the yeastreplication sequence element and an end homologous to a fragment (24)that bears the gene to be cloned, introduced as a result of the PCRprocess using the primer (15).

24 A DNA fragment [generated using the PCR process] that bears the geneto be cloned and an end homologous to the fragment (20) that bears theyeast selectable marker, introduced as a result of the PCR process usingthe primer (18).

26 An in vivo recombination intermediate between the overlapping DNAfragments (20), (22), and (24).

28 A recombinant plasmid, a product resulting from the in vivorecombination process, that bears the yeast selectable marker (11), theyeast replication sequence element (14), and the gene to be cloned (17).

FIG. 2 A multifragment cloning method based on homology introduced atboth ends of each fragment in a series.

30 A primer with a 3′ end homologous to one end of the top strand of theyeast marker gene (11) and with a 5′ end complementary to the 5′ end ofa primer (40).

32 A primer with a 3′ end complementary to the end of the top strand ofthe yeast marker gene (11), distal to the primer (30), and with a 5′ endcomplementary to the 5′ end of a primer (34).

34 A primer with a 3′ end homologous to one end of the top strand of theyeast replication sequence element (14) and with a 5′ end complementaryto the 5′ end of the primer (32).

36 A primer with a 3′ end complementary to the end of the top strand ofthe yeast replication sequence element (14), distal to the primer (34),and with a 5′ end complementary to the 5′ end of a primer (38).

38 A primer with a 3′ end homologous to one end of the top strand of thegene to be cloned (17) and with a 5′ end complementary to the 5′ end ofthe primer (36).

40 A primer with a 3′ end complementary to the end of the top strand ofthe gene to be cloned (17), distal to the primer (38), and with a 5′ endcomplementary to the 5′ end of the primer (30).

42 A DNA fragment [generated using the PCR process] that bears the yeastselectable marker and a 5′ end (a synthetic recombination target)derived from the 5′ end of the primer (30) as well as a 3′ end (a secondsynthetic recombination target) derived from the 5′ end of the primer(32).

44 A DNA fragment [generated using the PCR process] that bears the yeastreplication sequence element and a 5′ end (a synthetic recombinationtarget) derived from the 5′ end of the primer (34) as well as a 3′ end(a second synthetic recombination target) derived from the 5′ end of theprimer (36).

46 A DNA fragment [generated using the PCR process] that bears the geneto be cloned and a 5′ end (a synthetic recombination target) derivedfrom the 5′ end of the primer (38) as well as a 3′ end (a secondsynthetic recombination target) derived from the 5′ end of the primer(40).

48 An in vivo recombination intermediate between the overlapping DNAfragments (42), (44), and (46).

50 A recombinant plasmid, a product resulting from the in vivorecombination process, that bears the yeast selectable marker (11), theyeast replication sequence element (14), and the gene to be cloned (17).The yeast selectable marker (11) and the yeast replication sequenceelement (14) are linked via the recombination targets found at the 3′end of the DNA fragment (42) and the 5′ end of the DNA fragment (44).The yeast replication sequence element (14) and the gene to be cloned(17) are linked via the recombination targets found at the 3′ end of theDNA fragment (44) and the 5′ end of the DNA fragment (46). The gene tobe cloned (17) and the yeast selectable marker (11) are linked via therecombination targets found at the 3′ end of the DNA fragment (46) andthe 5′ end of the DNA fragment (42).

FIG. 3 A method of recombinational mapping of mutations.

52 A source of wild-type DNA of a gene of interest.

54 A source of mutant DNA of the gene of interest.

56 A primer homologous to the 5′ end of the top strand of the gene ofinterest, either the wild-type (52) or the mutant (54) allele.

58 A primer complementary to an internal region of the top strand of thegene of interest, either the wild-type (52) or the mutant (54) allele.

60 A fragment [that can be derived by PCR] from the wild-type gene (52)[that can be amplified by PCR using the primers (56) and (58)] thatextends from the sequence to which the primer (56) is homologous to thesequence to which the primer (58) is complementary.

62 A primer homologous to an internal region of the top strand of eitherthe wild-type (52) or the mutant (54) gene of interest and located 5′ tothe sequence to which the primer (58) is complementary.

64 A primer complementary to the 3′ end of the top strand of the gene ofinterest, either the wild-type (52) or the mutant (54) allele.

66 A fragment [that can be derived by PCR] from the wild-type gene (52)[that can be amplified by PCR using the primers (62) and (64)] thatextends from the sequence to which the primer (62) is homologous to thesequence to which the primer (64) is complementary.

68 A fragment [that can be derived by PCR] from the mutant gene (54)[that can be amplified by PCR using the primers (56) and (58)] thatextends from the sequence to which the primer (56) is homologous to thesequence to which the primer (58) is complementary.

70 A fragment [that can be derived by PCR] from the mutant gene (54)[that can be amplified by PCR using the primers (62) and (64)] thatextends from the sequence to which the primer (62) is homologous to thesequence to which the primer (64) is complementary.

72 A linear acceptor plasmid into which the DNA fragments (60 or 68) and(66 or 70) can be recombined in vivo.

FIG. 4 Recombinational mapping of mutations, a specific example.

80 A DNA fragment that includes a wild-type copy of the fliG gene, fusedbetween GAL4 and ADH1 DNA on a yeast expression vector, and that encodesa GAL4 DNA-binding domain-FliG fusion protein.

82 A DNA fragment that includes a mutant allele of the fliG gene, fusedbetween GAL4 and ADH1 DNA on a yeast expression vector, and that encodesa GAL4 DNA-binding domain-FliG fusion protein.

84 A DNA oligonucleotide primer (called “I”) that is homologous to thetop strand of a region of the DNA that encodes the GAL4 DNA-bindingdomain [contained within both the wild-type (80) and the mutant (82)GAL4-fliG gene fusions].

86 A DNA oligonucleotide primer (called “A”) that is homologous to thetop strand of a region of the DNA that encodes FliG [contained withinboth the wild-type (80) and the mutant (82) GAL4-fliG gene fusions]located 3′ of the primer (84) and 5′ of a primer (88).

88 A DNA oligonucleotide primer (called “C”) that is homologous to thetop strand of a region of the DNA that encodes FliG [contained withinboth the wild-type (80) and the mutant (82) GAL4-fliG gene fusions]located 3′ of the primer (86) and 5′ of the ADH1 terminator (a DNAsequence element that signals the termination of transcription).

90 A DNA oligonucleotide primer (called “B”) that is complementary to aregion of the top strand of the DNA that encodes FliG [contained withinboth the wild-type (80) and the mutant (82) GAL4-fliG gene fusions]located between the primers (86) and (88).

92 A DNA oligonucleotide primer (called “D”) that is complementary to aregion of the top strand of the DNA that encodes FliG [contained withinboth the wild-type (80) and the mutant (82) GAL4-fliG gene fusions]located between the primer (88) and the ADH1 terminator.

94 A DNA oligonucleotide primer (called “T”) that is complementary to aregion of the top strand of the ADH1 terminator [contained within boththe wild-type (80) and the mutant (82) GAL4-fliG gene fusions].

96 A DNA fragment amplified from the wild-type fliG (80) template usingPCR and the primers (84) and (90).

98 A DNA fragment amplified from the mutant fliG (82) template using PCRand the primers (86) and (94).

100 A DNA fragment amplified from the mutant fliG (82) template usingPCR and the primers (84) and (90).

102 A DNA fragment amplified from the wild-type fliG (80) template usingPCR and the primers (86) and (94).

104 A DNA fragment amplified from the wild-type fliG (80) template usingPCR and the primers (84) and (92).

106 A DNA fragment amplified from the mutant fliG (82) template usingPCR and the primers (88) and (94).

108 A DNA fragment amplified from the wild-type fliG (80) template usingPCR and the primers (84) and (92).

110 A DNA fragment amplified from the wild-type fliG (80) template usingPCR and the primers (88) and (94).

112 A linear receptor plasmid that has DNA homology at one end to GAL4DNA [the part of GAL4 present on DNA fragments (96), (100), (104), and(108)] and at the other end to ADH1 DNA [the part of ADH1 present on DNAfragments (98), (102), (106), and (110)]. The portion of GAL4 present onthe linear plasmid (112), when recombined with the overlapping GAL4 DNAon either fragment (96), (100), (104), or (108), is sufficient toreconstruct the GAL4 DNA-binding domain coding sequence. Similarly, theportion of ADH1 present on the linear plasmid (112), when recombinedwith the overlapping ADH1 DNA on fragment (98), (102), (106), or (110),is sufficient to reconstruct a complete ADH1 terminator element.

FIG. 5 Cassette-based cloning methods.

114 A primer homologous to a recombination element at the 5′ end of a“marker cassette.”

116 A primer complementary to a second unique recombination element atthe 3′ end of the “marker cassette.”

118 A primer with a 3′ end homologous to an oligo target sequence at the5′ end of a “replication cassette” and with a 5′ end homologous to therecombination element at the 3′ end of the “marker cassette.”

120 A primer complementary to a third unique recombination element atthe 3′ end of the “replication cassette.”

122 A primer with a 3′ end homologous to the 5′ end of a gene to becloned and with a 5′ end homologous to the recombination element at the3′ end of the “replication cassette.”

124 A primer with a 3′ end complementary to the 3′ end of the gene to becloned and with a 5′ end complementary to the recombination element atthe 5′ end of the “marker cassette.”

126 A source of a marker cassette, for example, a plasmid or a linearDNA fragment, that contains a yeast selectable marker bounded by the 5′marker recombination element and the 3′ marker recombination element. Byusing different yeast genes separately cloned into the same or similarvectors, interchangeable cassettes of different selectable markers canbe generated.

128 A source of a replication cassette, for example, a plasmid or alinear DNA fragment, that contains a yeast replication element boundedby the 5′ oligo target sequence and the 3′ replication recombinationelement. By using different yeast replication elements separately clonedinto the same or similar vectors, interchangeable cassettes of differentrecombination elements can be generated.

130 A source of the gene to be cloned.

132 A linear fragment that contains the yeast selectable marker boundedby the 5′ marker recombination element and the 3′ marker recombinationelement. This fragment is generated by PCR using the marker cassettesource (126) as template and the primers (114) and (116).

134 A linear fragment that contains the yeast replication elementbounded by the 5′ replication recombination element (homologous to the3′ marker recombination element) and by the 3′ replication recombinationelement. This fragment is generated by PCR using the replicationcassette source (128) as template and the primers (118) and (120).

136 A linear fragment that contains the gene to be cloned bounded by a5′ recombination element (homologous to the 3′ replication recombinationelement) and a 3′ recombination element (homologous to the 5′ markerrecombination element). This fragment is generated by PCR using the genesource (130) as template and the primers (122) and (124).

138 An in vivo recombination intermediate between the overlapping DNAfragments (132), (134), and (136).

140 A recombinant plasmid, a product of homologous recombination,generated in vivo.

FIG. 6 Directed-evolution based on multifragment cloning.

141 A wild-type evolution cassette; a source of a wild-type gene to beevolved, having at its 3′ end a region homologous to one end of a gappedvector (168) and at its 5′ end a region homologous to the other end of agapped vector (168).

142 A primer homologous to the 5′ end of a DNA (evolution cassette) tobe evolved and also homologous to one end of a linearized gapped vector(168).

143 A mutant evolution cassette; a source of a mutant gene bearingmutation 1 (isolated in a primary selection for improved gene function;one of many such mutations that are present in a pool of selectedgenes).

144 A primer complementary to a region near the 3′ end of the DNA(evolution cassette) to be evolved, but not within a terminal region ofhomology to a linearized gapped vector (168).

145 A second mutant evolution cassette; a source of a mutant genebearing mutation 2 (isolated in a primary selection for improved genefunction; one of many such mutations that are present in a pool ofselected genes).

146 A primer homologous to a region near the 5′ end of a DNA (evolutioncassette) to be evolved, but not within a terminal region of homology toa linearized gapped vector (168).

148 A primer complementary to the 3′ end of a DNA (evolution cassette)to be evolved, located within a region that is homologous to the otherend of a linearized gapped vector (168).

156 A DNA fragment amplified by PCR from the wild-type DNA source (141)using the primers (142) and (144).

158 A DNA fragment amplified by PCR from the wild-type DNA source (141)using the primers (146) and (148).

160 A DNA fragment amplified by PCR from the mutation 1 DNA source (143)using the primers (142) and (144).

162 A DNA fragment amplified by PCR from the mutation 1 DNA source (143)using the primers (146) and (148).

164 A DNA fragment amplified by PCR from the mutation 2 DNA source (145)using the primers (142) and (144).

166 A DNA fragment amplified by PCR from the mutation 2 DNA source (145)using the primers (146) and (148).

168 A linearized and gapped acceptor plasmid with a DNA replicationorigin and a marker that can be selected for in yeast.

170 A recombination intermediate between the gapped acceptor plasmid(168), the wild-type fragment (156), and the overlapping wild-typefragment (158). Recombination in vivo regenerates the wild-type gene, ona vector.

172 A recombination intermediate between the gapped acceptor plasmid(168), the DNA fragment (160) containing mutation 1, and the DNAfragment (162) also containing mutation 1. Recombination in vivoregenerates mutation 1, on a vector.

174 A recombination intermediate between the gapped acceptor plasmid(168), the DNA fragment (160) containing mutation 1, and the DNAfragment (166) containing mutation 2, regenerating a mutation 1-bearingvector.

176 A recombination intermediate between the gapped acceptor plasmid(168), the DNA fragment (160) containing mutation 1, and the DNAfragment (166) containing mutation 2, regenerating a mutation 1-mutation2 double-mutant-bearing vector.

178 A recombination intermediate between the gapped acceptor plasmid(168), the DNA fragment (160) containing mutation 1, and the DNAfragment (166) containing mutation 2, regenerating a mutation 2-bearingvector.

180 A recombination intermediate between the gapped acceptor plasmid(168), the DNA fragment (164) containing mutation 2, and the DNAfragment (162) containing mutation 1, regenerating a mutation 1-bearingvector.

182 A recombination intermediate between the gapped acceptor plasmid(168), the DNA fragment (164) containing mutation 2, and the DNAfragment (162) containing mutation 1, regenerating a wild-type-bearingvector.

184 A recombination intermediate between the gapped acceptor plasmid(168), the DNA fragment (164) containing mutation 2, and the DNAfragment (162) containing mutation 1, regenerating a mutation 2-bearingvector.

186 A recombination intermediate between the gapped acceptor plasmid(168), the DNA fragment (164) containing mutation 2, and the DNAfragment (166) also containing mutation 2, regenerating a mutation2-bearing vector.

DETAILED DESCRIPTION OF THE INVENTION Description—FIGS. 1 to 6

FIG. 1 A multifragment cloning method based on homology introduced atone end of each fragment in a series.

A method of cloning DNA is described. This method is based on theability to create a series of overlapping DNA fragments by introducingat one end of each DNA fragment a sequence element homologous to thenext DNA fragment in the series (see FIG. 1).

This method can be used to construct vectors that stably transform yeastand that contain only endogenous yeast sequences.

This method requires a host with an efficient and highly accuraterecombination system. We have found that the yeast Saccharomycescerevisiae serves as a suitable transformation host for this method. Wehave tested many strains of S. cerevisiae and all have served assuitable hosts. We have tested other possible transformation hosts,including several strains of E. coli, but so far we have not identifiedany other hosts suitable for use in this method.

The yeast vector to be constructed is assembled from three (or more)component parts. Minimally these parts consist of 1) a yeast selectablemarker (11), 2) a yeast DNA replication element (14), and 3) a gene tobe cloned (17). Methods to construct vectors suitable for yeasttransformation from these component parts are described.

FIG. 1, DNA fragment (11)—A DNA fragment containing a yeast selectablemarker. Typically this fragment will serve as a template foramplification by PCR (using DNA primers 10 and 12) from a yeast vectorcontaining the selectable marker. Alternately, a linear fragmentcontaining the selectable marker could be used as a template.

FIG. 1, DNA fragment (14)—A DNA fragment containing a yeast DNA originof replication (ARS—autonomously replicating sequence) element.Optionally this fragment can contain, in addition to the DNA replicationorigin, a stability element such as a centromere or a 2μ STAB sequence.Typically this fragment will serve as a template for amplification byPCR (using DNA primers 13 and 15) from a yeast vector containing a yeastreplication origin (and optionally a stability element). Alternately, alinear fragment containing an ARS and optionally a STAB element could beused as a template.

FIG. 1, DNA fragment (17)—A DNA fragment containing the gene to becloned. Typically this fragment will serve as a template foramplification by PCR (using DNA primers 16 and 18) from a cloning vectorcontaining the gene. Alternately, a linear fragment containing the genecould be used as a template.

FIG. 1, DNA primer (10). Primer homologous to one end of the top strandof the yeast marker gene (11), to be used in combination with primer(12) to amplify the DNA substrate (11) using the PCR process.

FIG. 1, DNA primer (12). Primer with a 3′ end homologous to the otherend of the bottom strand of the yeast marker gene (11) and with a 5′ endcomplementary to primer (13).

FIG. 1, DNA primer (13). Primer homologous to one end of the top strandof the yeast replication sequence element (14), to be used incombination with primer (15) to amplify the DNA substrate (14) using thePCR process.

FIG. 1, DNA primer (15). Primer with a 3′ end homologous to the otherend of the bottom strand of the yeast replication element (14) and witha 5′ end complementary to primer (16).

FIG. 1, DNA primer (16). Primer homologous to one end of the top strandof the gene to be cloned (17).

FIG. 1, DNA primer (18). Primer with a 3′ end complementary to the otherend of the top strand of the gene to be cloned (17) and with a 5′ endcomplementary to primer (10).

Primer (12) and primer (10) are used in the PCR process to amplify alinear DNA using the yeast marker gene (11) as a substrate. The fragmentgenerated (20) bears the yeast selectable marker and an end homologousto the fragment (22) that bears the yeast replication element,introduced as a result of the PCR process using the primer (12).

Primer (13) and primer (15) are used in the PCR process to amplify alinear DNA using the yeast DNA replication element (14) as a substrate.The fragment generated (22) bears the yeast replication element and anend homologous to the fragment (24) that bears the gene to be cloned,introduced as a result of the PCR process using the primer (15).

Primer (16) and primer (18) are used in the PCR process to amplify alinear DNA using the gene to be cloned (17) as a substrate. The fragmentgenerated (24) bears the gene to be cloned and an end homologous to thefragment (20) that bears the yeast selectable marker, introduced as aresult of the PCR process using the primer (18).

DNA fragments (20), (22), and (24) are mixed an used to transform theyeast S. cerevisiae using standard procedures. The transformants aresubjected to the appropriate selection for the selectable marker carriedon the DNA element (11).

FIG. 1, (26). Recombination intermediate that occurs in vivo.

FIG. 1, (28). Following recombination, a circular plasmid results fromthe in vivo recombination process.

The transformed yeast can be examined directly for phenotypes that mightbe associated with the cloned gene, for example a growth phenotype or avisual enzymatic activity.

DNA can be isolated from individual colonies (or from cultures derivedfrom individual colonies) to verify the plasmid construction and/or totest in other ways (for example, to transfer the plasmid into a newyeast strain for subsequent analysis).

FIG. 2 A multifragment cloning method based on homology introduced atboth ends of each fragment in a series.

Another method of cloning DNA is described. This method is based on theability to create a series of overlapping DNA fragments by introducingat one end of each DNA fragment a sequence element homologous to oneadded to the previous DNA fragment in the series, and by introducing atthe other end of each DNA fragment a different sequence element alsoadded to the next DNA fragment in the series (see FIG. 2).

This method can be used to construct vectors that contain specificsequence information separating the other DNA elements of the plasmid(as in FIG. 2, between the selectable marker, the yeast replicationsequence, and the gene to be cloned). For example, by designing theprimer (30) and the primer (40) so that their 5′ portions arecomplementary and each contain the same restriction enzyme recognitionsequence, a specific site can be introduced between the selectablemarker and the gene to be cloned. Likewise, the primer pairs (32 and 34)and (36 and 38) can be designed to each introduce (via theircomplementary 5′ ends) a unique DNA sequence separating the plasmidparts that they join.

This method requires a host with an efficient and highly accuraterecombination system. We have found the yeast S. cerevisiae serves as asuitable transformation host for this method. We have tested manystrains of S. cerevisiae and all have served as suitable hosts. We havetested other possible transformation hosts, including several strains ofE. coli, but have not identified any other hosts suitable for use inthis method.

The yeast vector to be constructed is assembled (see FIG. 2) from three(or more) component parts. Minimally these parts consist of 1) a yeastselectable marker (11), 2) a yeast DNA replication element (14), and 3)a yeast gene to be cloned (17). Methods to construct vectors suitablefor yeast transformation from these component parts are described.

FIG. 2, DNA fragment (11)—A DNA fragment containing a yeast selectablemarker. Typically this fragment will serve as a template foramplification by PCR (using DNA primers 30 and 32) from a yeast vectorcontaining the selectable marker. Alternately, a linear fragmentcontaining the selectable marker could be used as a template.

FIG. 2, DNA fragment (14)—A DNA fragment containing a yeast DNA originof replication (ARS—autonomously replicating sequence) element.Optionally this fragment can contain, in addition to the DNA replicationorigin, a stability element such as a centromere or a 2μ STAB sequence.Typically this fragment will serve as a template for amplification byPCR (using DNA primers 34 and 36) from a yeast vector containing a yeastreplication origin (and optionally a stability element). Alternately, alinear fragment containing an ARS and optionally a STAB element could beused as a template.

FIG. 2, DNA fragment (17)—A DNA fragment containing the gene to becloned. Typically this fragment will serve as a template foramplification by PCR (using DNA primers 38 and 40) from a cloning vectorcontaining the gene. Alternately, a linear fragment containing the genecould be used as a template.

FIG. 2, DNA primer (30)—A primer with a 3′ end homologous to one end ofthe top strand of the yeast marker gene (11) and with a 5′ endcomplementary to the 5′ end of primer (40), to be used in conjunctionwith DNA primer (32) to amplify DNA containing the selectable marker(11) to generate fragment (42).

FIG. 2, DNA primer (32). Primer with a 3′ end homologous to the otherend of the bottom strand of the yeast marker gene (11) and with a 5′ endcomplementary to the 5′ end of primer (34).

FIG. 2, DNA primer (34). Primer with a 3′ end homologous to one end ofthe top strand of a yeast replication sequence element (14) and with a5′ end complementary to the 5′ end of primer (32).

FIG. 2, DNA primer (36). Primer with a 3′ end homologous to the otherend of the bottom strand of the yeast replication element (14) and witha 5′ end complementary to the 5′ end of primer (38).

FIG. 2, DNA primer (38). Primer with a 3′ end homologous to one end ofthe top strand of the gene to be cloned (17) and with a 5′ endcomplementary to the 5′ end of primer (36).

FIG. 2, DNA primer (40). Primer with a 3′ end homologous to the topstrand of the gene to be cloned (17) and with a 5′ end complementary tothe 5′ end of primer (30).

FIG. 2, DNA fragment (42). DNA fragment generated using the PCR processbearing the yeast selectable marker and a 5′ end (a syntheticrecombination target) derived from the 5′ end of the primer (30) as wellas a 3′ end (a second synthetic recombination target) derived from the5′ end of the primer (32).

FIG. 2, DNA fragment (44). DNA fragment generated using the PCR processbearing the yeast replication element and a 5′ end (a syntheticrecombination target) derived from the 5′ end of the primer (34) as wellas a 3′ end (a second synthetic recombination target) derived from the5′ end of the primer (36).

FIG. 2, DNA fragment (46). DNA fragment generated using the PCR processbearing the gene to be cloned and a 5′ end (a synthetic recombinationtarget) derived from the 5′ end of the primer (38) as well as a 3′ end(a second synthetic recombination target) derived from the 5′ end of theprimer (40).

DNA fragments (42), (44), and (46) are mixed and used to transform theyeast S. cerevisiae using standard procedures. The transformants aresubjected to the appropriate selection for the selectable marker carriedon the DNA element (11).

FIG. 2, (48). Recombination intermediate that occurs in vivo.

FIG. 2, (50) Following recombination, a circular plasmid results fromthe in vivo recombination process, bearing the yeast selectable marker(11), the yeast replication sequence (14), and the gene to be cloned(17). The yeast selectable marker (11) and the yeast replicationsequence (14) recombine at the synthetic recombination targets found atthe 3′ end of the DNA fragment (42) and the 5′ end of the DNA fragment(44) and thus are linked. The yeast replication sequence (14) and thegene to be cloned (17) recombine at the synthetic recombination targetsfound at the 3′ end of the DNA fragment (44) and the 5′ end of the DNAfragment (46) and thus are linked. The gene to be cloned (17) and theyeast selectable marker (11) recombine at the synthetic recombinationtargets found at the 3′ end of the DNA fragment (46) and the 5′ end ofthe DNA fragment (42) and thus are linked. These three recombinationevents together can create a circular plasmid that contains a DNAreplication sequence (14) (ARS) as well as a yeast selectable marker(11) and the gene to be cloned (17). Thus, selection for the yeastmarker (and concomitantly the DNA replication origin (14)) selects forplasmids that have undergone the “3-way recombination” and so containthe gene to be cloned.

The transformed yeast that bear recombinant circular plasmids thatinclude the DNA for the selectable marker (11) grow to form colonies ona yeast transformation plate of the appropriate nutritional selection.For example, when using the HIS3 gene to complement a his3⁻ mutation,one would select for the proper yeast on plates lacking histidine.

These transformed yeast can be examined directly for phenotypes thatmight be associated with the cloned gene, for example a growth phenotypeor a visual enzymatic activity.

DNA can be isolated from individual colonies (or from cultures derivedfrom single colonies) to verify the plasmid construction and/or to testin other ways (for example, to transfer the plasmid into a new yeaststrain for subsequent analysis).

FIG. 3 A method of recombinational mapping of mutations.

Mapping mutations using multifragment in vivo cloning.

A method is presented for mapping the location of a mutation within agene phenotypically expressed in yeast. We have developed this method torapidly map mutations that disrupt an interaction identified using thetwo-hybrid system originally described by Fields, S. & Song, O.-k.(Nature 340: 245-246, 1989). This method can be used to map the locationof mutations within genes that are expressed in yeast so long as thegene function can be identified by some phenotypic assay. Thus genesfrom yeast or other organisms can be studied if they are associated witha phenotype such as growth (ie. a nutritional requirement, orcomplementation of an essential function), an assayable enzymaticactivity, or an interaction that is coupled to such an identifiablephenotype.

Wild-type and mutant genes would typically be contained on plasmids thatreplicate and express in yeast, but might also be from plasmids that donot replicate in yeast, or from linear DNA fragments. These plasmids (orlinear fragments derived from them), are shown in FIG. 3. A wild-typegene (52), and a mutant gene (54) (the asterisk indicates the locationof a mutation that will be mapped in an example) serve as substrates forPCR using the primer pairs (56 and 58) and (62 and 64). Primers (58) and(62) are designed so that either they overlap, or so that they amplifyfragments which overlap. We have found that an overlap with as few as 17nucleotides is sufficient.

In a first container, primer (56) and primer (58) (see FIG. 3) are usedto amplify by PCR the left side of the WT gene (52) to generate DNAfragment (60). It is important to use a polymerase with low error ratessuch as Pfu DNA polymerase, so that a minimal number of errors areintroduced that might otherwise obscure the mapping data.

In a second container, primer (62) and primer (64) (see FIG. 3) are usedto amplify by PCR the right side of the WT gene (52) to generate DNAfragment (66). It is important to use a polymerase with low error ratessuch as Pfu DNA polymerase, so that a minimal number of errors areintroduced that might otherwise obscure the mapping data.

In a third container, primer (56) and primer (58) (see FIG. 3) are usedto amplify by PCR the left side of the mutant gene (54) to generate DNAfragment (68). It is important to use a polymerase with low error ratessuch as Pfu DNA polymerase, so that a minimal number of errors areintroduced that might otherwise obscure the mapping data.

In a fourth container, primer (62) and primer (64) (see FIG. 3) are usedto amplify by PCR the right side of the mutant gene (54) to generate DNAfragment (70). It is important to use a polymerase with low error ratessuch as Pfu DNA polymerase, so that a minimal number of errors areintroduced that might otherwise obscure the mapping data.

Primers (58) and (62) (see FIG. 3) are designed so that the 3′ end ofthe left fragment (60) and the 5′ end of the right fragment (66) containa region of overlap, or homology. (In FIG. 3, the DNA fragment pairs(68) and (70), (60) and (70) and finally (68) and (66) have the sameoverlap as the pair (60) and (66). We have used overlaps as small as 17bp.

In a fifth container, a gapped acceptor vector (72) is generated bycutting a plasmid (that can replicate and be selected for in yeast) witha suitable restriction enzyme. A linearized (gapped) plasmid is unableto replicate in yeast. The gapped plasmid (72) contains homology (nearone end of the gap) with the 5′ end of the left fragment (60) and alsocontains homology (near the other end of the gap) with the 3′ end of theright fragment (66). Alternately, a linearized plasmid with the samestructure can be generated using PCR; however, due to the size of manyof the yeast plasmids used to construct gapped acceptor vectors, the PCRreaction is often inefficient.

In a sixth container, an aliquot of DNA fragment (60) amplified by thePCR reaction from container 1 is mixed with an aliquot of DNA fragment(66) amplified by the PCR reaction from container 2. To this mixture isadded gapped acceptor plasmid (72) from container 5. This mixture isused to transform yeast by standard techniques, selecting for the markerpresent on the gapped acceptor plasmid. The gapped acceptor plasmid andthe left and right fragments undergo a “3-way” recombination to generatea circular plasmid (see FIG. 3). The circular plasmid generated willreplicate and can be selected for in yeast using standard methods. Thistransformation represents a wild-type reconstruction.

In a seventh container, an aliquot of DNA fragment (60) amplified by thePCR reaction from container 1 is mixed with an aliquot of DNA fragment(70) amplified by the PCR reaction from container 4. To this mixture isadded gapped acceptor plasmid (72) from container 5. This mixture isused to transform yeast by standard techniques, selecting for the markerpresent on the gapped acceptor plasmid. The gapped acceptor plasmid andthe left and right fragments undergo a “3-way” recombination to generatea circular plasmid. The circular plasmid generated will replicate andcan be selected for in yeast using standard methods. This transformationtests the location of the mutation; it will regenerate a phenotypicallywild-type plasmid if the mutation had been in fragment (68) (asdiagramed in FIG. 3), and a phenotypically mutant plasmid if themutation had been in fragment (70).

In an eighth container, an aliquot of DNA fragment (68) amplified by thePCR reaction from container 3 is mixed with an aliquot of DNA fragment(66) amplified by the PCR reaction from container 2. To this mixture isadded gapped acceptor plasmid (72) from container 5. This mixture isused to transform yeast by standard techniques, selecting for the markerpresent on the gapped acceptor plasmid. The gapped acceptor plasmid (72)and the left (68) and right (66) fragments undergo a “3-way”recombination to generate a circular plasmid. The circular plasmidgenerated will replicate and can be selected for in yeast using standardmethods. This transformation tests the location of the mutation; it willregenerate a phenotypically wild-type plasmid if the mutation had beenin the right fragment (70), and a phenotypically mutant plasmid if themutation had been in the left fragment (68) (as diagramed in FIG. 3).

FIG. 5 Cassette-based cloning methods.

A method to construct plasmids in yeast from overlapping DNA cassettesusing multifragment in vivo recombination.

Sources of yeast marker gene(s), yeast DNA replication sequences(s), anda gene to be cloned are constructed by standard cloning techniques, orobtained by other means.

A source of the marker gene (126) (see FIG. 5) with a sequence elementhomologous to the primer (114) and with another sequence elementcomplementary to the primer sequence (116) surrounding a gene that canbe selected for in yeast (a yeast marker). These sequence elements canserve both as priming sites for PCR amplification and as recombinationtargets in subsequent steps. In one preferred embodiment, a series ofdifferent (yeast marker) fragments are constructed, each with adifferent selectable marker, but surrounded by the same PCR primingsites/recombination targets. For example, this can be achieved bycloning a series of yeast marker genes into a common plasmid at adefined location on the plasmid, for example into a polylinker. Thesecassettes serve as interchangeable sources of a yeast marker gene insubsequent recombination steps.

Source of the replication cassette (128) (see FIG. 5) with a sequenceelement homologous to the 3′ end of primer (118) and another sequenceelement complementary to the primer sequence (120) surrounding a yeastDNA replication origin (or ARS for Autonomously Replicating Sequence)and optionally a centromere or other stability sequence such as the 2μSTAB element. These sequence elements can serve as priming sites for PCRamplification. The sequence element complementary to the primer sequence(120) can serve as a recombination target in subsequent steps. In onepreferred embodiment, a series of different (DNA replication) fragmentsare constructed, each with a different yeast DNA replication originand/or stability element flanked by the same PCR priming sites. Forexample, this can be achieved by cloning a series of DNA replicationelements into a common plasmid at a defined location on the plasmid, forexample into a polylinker. These cassettes serve as interchangeablesources of a yeast replication element in subsequent recombinationsteps.

A source of the gene to be cloned (130) (see FIG. 5). This source couldbe a plasmid containing the gene, a linear fragment containing the gene,genomic DNA containing the gene, or a cDNA mixture containing the gene.

One method to construct the linear DNA fragment (132) with arecombination element homologous to the primer (114) and anotherrecombination element complementary to the primer sequence (116):

Primer (114) (see FIG. 5) is homologous to the 5′ end of the top strandof (126) which serves as a source of the yeast selectable marker gene.Primer (116) is complementary to the 3′ end of the top strand of (126).Primers (114) and (116) are then used as primers in a PCR amplificationusing DNA fragment (126) as a substrate to amplify DNA fragment (132).

One method to construct the linear DNA fragment (134) with arecombination element complementary to the primer (116) and anotherrecombination element complementary to the primer sequence (120): Primer(118) (FIG. 5) was designed such that the 5′ end is complementary toprimer (116) and the 3′ end of primer (118) is homologous to thesequence at the 5′ end of the top strand of the yeast replicationcassette (128). Primer (120) (FIG. 5) is complementary to the sequenceat the 3′ end of the top strand of the yeast replication cassette (128).Primers (118) and (120) are then used as primers in a PCR amplificationusing the replication cassette (128) as a substrate (either from aplasmid with this sequence element, or from a linear fragment with thiselement) to generate the linear fragment (134).

One method to construct the linear DNA fragment (136) with arecombination element complementary to the primer (114) and anotherrecombination element complementary to the primer sequence (120):

Primer (122) (FIG. 5) was designed such that the 5′ end of primer (122)is complementary to primer (120) and the 3′ end of primer (122) ishomologous to the sequence at the 5′ end of the top strand of the geneto be cloned on DNA element (130). Primer (124) (FIG. 5) was designed sothat the 5′ end of primer (124) is complementary to primer (114) and the3′ end of primer (124) is complementary to the 3′ end of the top strandof the gene to be cloned on DNA element (130). Primers (122) and (124)are then used as primers in a PCR amplification using the source (forexample, a plasmid of a linear fragment) of the gene to be cloned (130),as a substrate to generate DNA fragment (136).

The recombination targets [the sequence element homologous to primer(114), the sequence element complementary to primer (116), and thesequence element complementary to primer (120)] can be syntheticsequences (not derived from functional yeast sequences) designed solelyas recombination targets. Alternately, these recombination sequences[the sequence element homologous to primer (114), the sequence elementcomplementary to primer (116), and the sequence element complementary toprimer (120)] could be functional elements such as promoter (UAS) orterminator elements. Or, alternately, these recombination sequences [thesequence element homologous to primer (114), the sequence elementcomplementary to primer (116), and the sequence element complementary toprimer (120)] might be designed to introduce restriction sites or otherspecific DNA sequences that might be useful in conventional cloning orDNA amplifications.

DNA Fragments (132), (134), and (136) are mixed and used to transformyeast, selecting for the marker on DNA fragment (132).

These fragments recombine in yeast, represented by the in vivorecombination intermediate (138) (see FIG. 5). The sequence elementcomplementary to primer (116) at one end of DNA fragment (132)recombines with the sequence element complementary to primer (116) atone end of DNA fragment (134). The sequence element complementary toprimer (120) at one end of DNA fragment (134) recombines with thesequence element complementary to primer (120) at one end of DNAfragment (136). The sequence element homologous to primer (114) at oneend of DNA fragment (136) recombines with the sequence elementhomologous to primer (114) at one end of DNA fragment (132).

These three recombination events join together the three overlapping DNAfragments (132), (134), and (136) into a circular plasmid (140) thatcontains a DNA replication sequence (128), a yeast selectable marker(126), and the gene to be cloned (130). Any circular plasmids formedthat do not contain both the DNA replication sequences (a yeast ARSelement) and the yeast selectable marker gene will not be replicated andmaintained during selection for the yeast marker. The homologousrecombination process in yeast is highly efficient and the majority ofthe plasmids that survive the transformation and selection process arefound to have incorporated the other fragment (fragments 136 thisexample) that were designed to create a circular DNA plasmid moleculeupon recombination in vivo.

The transformed yeast cells that bear recombinant circular plasmids growto form colonies on a yeast transformation plate that selects for theyeast marker derived from DNA fragment (132).

Isolates of plasmid DNA with the correct structure can be identified andisolated by preparing DNA from several single yeast colonies (orcultures derived from single colonies) and examining this DNA directly(by restriction enzyme analysis or PCR analysis) to confirm that theplasmid construction was successful. This DNA can be used to transformE. coli in order to generate large quantities of plasmid DNA.

The transformed yeast can be examined directly for phenotypes that mightbe caused by the cloned gene; for example a growth phenotype.

One possible modification of this method would be the construction ofmarker cassettes and replication cassettes that contain telomeres at theends. In this case the selection would not be the formation of acircular plasmid that was able to replicate, but rather the formation ofa linear plasmid that could replicate in yeast. In this case one wouldjoin a telomere+marker cassette and a telomere+replication cassette viaan intermediate DNA fragment containing at one end a region homologousto the telomere-distal end of the telomere+marker cassette and at theother end a region homologous to the telomere distal end of thetelomere+replication cassette.

FIG. 6. Directed-evolution based on multifragment cloning.

A method of accelerated in vivo evolution (mutagenesis in vitro,followed by sexual in vivo recombination that results in thereassortment of multiple mutations, followed by genetic selection foradvantageous combinations of the multiple mutations).

A method is described to generate and identify combinations of multiplemutations that alter a function (specificity, activity, affinity, etc.)of an enzyme (or other protein or DNA or RNA molecule). In particular,multifragment cloning in vivo provides a means of recombining multiplemutations (that are each alone responsible for a small functionalchange) into compositie multimutants (exhibiting a composite phenotype),thus allowing the efficient creation of new macromolecules withsignificantly modified function.

An “evolution cassette” (141) (FIG. 6), including the gene to bemutated, is mutagenized using standard techniques, or by mutagenic PCR.

The “evolution cassette” (141) (FIG. 6) includes regions that flank thegene to be mutated and that are homologous with the ends of the gappedrecipient vector (168). Primer (142) is homologous to the 5′ end of thetop strand of the evolution cassette (141). Primer (148) iscomplementary to the 3′ end of the top strand of the evolution cassette(141). Primer (146) is homologous to the 5′ end of the top strand of theregion of the “evolution cassette” containing the region of the gene tobe mutagenized and evolved. Primers (142) and (146) should not have anyoverlap (or homology). Primer (144) is complementary to the 3′ end ofthe top strand of the region of the “evolution cassette” containing theregion of the gene to be mutagenized and evolved. Primers (144) and(148) should not have any overlap (or homology).

The “evolution cassette” (141) may be mutagenized using standardtechniques. Then a pool of linear mutagenized “evolution cassette” DNAfragments is generated by PCR amplification of the mutagenized“evolution cassette” DNA using primer (142) and primer (148) and athermostable DNA polymerase with a low error rate such as Pfu DNApolymerase.

Alternatively, the “evolution cassette” may be mutagenized and amplifiedin one step by mutagenic PCR. A pool of mutagenized “evolution cassette”DNA fragments is generated by PCR amplification of wild-type DNA (141)using primer (142) and primer (148) and an error prone thermostable DNApolymerase such as Taq DNA polymerase.

The gapped yeast plasmid (168) is derived from a yeast plasmid that canreplicate and be selected for in yeast. This plasmid can be generatedeither by linearizing a plasmid with an appropriate restriction enzyme(or pair of enzymes) or by using the PCR process to amplify the regionof the plasmid. Such a plasmid contains a yeast replication sequence(ARS—autonomously replicating sequence) and a yeast marker gene that canbe selected in yeast following transformation (such as a nutritionalmarker). In addition, this vector contains a recombination sequenceelement that is also present at the 5′ end of the “evolution cassette”DNA fragment (the 5′ recombination sequence) and a recombinationsequence element that is also present at the 3′ end of the “evolutioncassette” DNA fragment (the 3′ recombination sequence). The polarity ofthe 5′ and 3′ recombination sequence elements is the same as in the“evolution cassette”.

The pool of mutagenized “evolution cassette” DNA (generated by either ofthe above two procedures) is mixed with the gapped recipient vector(168) and used to transform yeast using standard techniques, selectingfor the yeast marker present on the gapped plasmid. The gapped acceptorplasmid and the mutagenized “evolution cassette” undergo recombinationin vivo to generate a circular plasmid. The circular plasmid generatedwill replicate and can be selected for in yeast using standard methods.Yeast containing plasmids which have incorporated the “evolutioncassette” DNA will grow into colonies on transformation plates whenselection is maintained for the yeast marker present on the gappedplasmid.

Yeast bearing mutant plasmids can be identified by an appropriate screenor selection for the desired mutations. Yeast bearing plasmids withmutations conferring upon the “gene to be mutated” (an enzyme or otherprotein, DNA or RNA molecule) an intermediate (or small) change inspecificity and/or activity and/or affinity are thus identified. Yeastbearing mutant plasmids with the desired mutations are pooled and theirplasmid DNA is isolated to be used in the following steps of in vivorecombination.

In one container, DNA is amplified using the pool of mutant plasmid DNAsas a substrate for a PCR reaction using primers (142) and (144), thusgenerating a pool of mutant DNA fragments with a recombination targetsequence 5′ of the gene to be mutated. DNA fragment (160) is such afragment derived from one particular mutation present in this pool ofmutated DNA. DNA fragment (164) is another such a fragment derived froma different mutant DNA present in this pool of mutated DNA. DNA fragment(156) is another such a fragment derived from a wild-type DNA present inthis pool of mutated DNA.

In a second container, DNA is amplified using the pool of mutant plasmidDNAs as a substrate for a PCR reaction using primers (146) and (148),thus generating a pool of mutant DNA fragments with a recombinationtarget sequence 3′ of the gene to be mutated. DNA fragment (162) is sucha DNA fragment derived from one particular mutation present in this poolof mutated DNA. DNA fragment (166) is another such a fragment derivedfrom a different mutant DNA present in this pool of mutated DNA. DNAfragment (158) is another such a fragment derived from a wild-type DNApresent in this pool of mutated DNA.

The pool of “5′ recombination sequence+gene to be mutated” DNA (similarto the wild-type fragment (156), only this is a pool of various mutantDNAs) and the pool of “gene to be mutated+3′ recombination sequence” DNA(similar to the wild-type fragment (158), only this is a pool of variousmutant DNAs) are mixed with the gapped recipient vector and used totransform yeast using standard techniques, selecting for the yeastmarker present on the gapped plasmid. The gapped acceptor plasmid, the“5′ recombination sequence+gene to be mutated” DNA, and the “gene to bemutated+3′ recombination sequence” DNA undergo a 3-way recombination invivo to generate a circular plasmid. Recombination intermediate (170)represents the recombination between the gapped vector (168) and the twowild-type DNA fragments (156) and (158), regenerating a wild-type copyof the gene on a plasmid. Recombination intermediate (172) representsthe recombination between the gapped vector (168) and the mutant 1 DNAfragments (160) and (162), regenerating a mutant copy of the gene(bearing mutation 1) on a plasmid. Recombination intermediate (186)represents the recombination between the gapped vector (168) and themutant 2 DNA fragments (164) and (166), regenerating a mutant copy ofthe gene (bearing mutation 2) on a plasmid.

Intermediates (174), (176), (178), (180), (182), and (184) represent themany different recombination intermediates that can be formed betweenthe mutant 1 DNA fragment (160) and the mutant 2 DNA fragment (166) orbetween the mutant 2 DNA fragment (162) and the mutant 1 DNA fragment(164).

Recombination intermediates (174) and (180) regenerate a mutation1-bearing DNA.

Recombination intermediate (182) regenerates a wild-type DNA.

Recombination intermediates (178) and (184) regenerate a mutation2-bearing DNA.

Recombination intermediate (176) is of particular interest, because itrepresents the class of recombinants that have successfully recombinedtwo mutant DNAs into one DNA bearing both mutations.

The circular plasmids generated will replicate and can be selected forin yeast using standard methods. Yeast containing plasmids with themutated gene of interest will grow into colonies on transformationplates when selection is maintained for the yeast marker present on thegapped plasmid.

Among the double mutants represented by the recombination intermediate(176), one expects to find double mutants that have an improvedfunction. Such evolved genes can be identified using a suitablephenotypic screen or selection.

This process of recombination and selection can be repeated multipletimes, allowing for a combinatorial search of multiple mutants byrecombining and screening pools of preselected mutants, thus avoidingthe impossibly large task of separately creating and testing allpossible combinations of multiple mutations.

Yeast bearing mutant plasmids are identified, and plasmid DNA isisolated from individuals to obtain the evolved genes.

The present invention can be illustrated by the following non-limitingexample.

Mapping by multifragment cloning in vivo

An efficient method for mapping mutations is described in which hybridgenes, derived partly from mutant and partly from wild-type DNA, areobtained in vivo by homologous recombination of multiple fragments. Therecombinants are formed in a strain in which their phenotypes areimmediately apparent. This method was developed to identify changes thatdisrupt protein—protein interactions demonstrable by the two-hybridsystem in yeast. However, it can be extended to any system whererecombination is possible, provided an assay is available to distinguishbetween mutant and wild-type phenotypes.

Traditional cloning methods are often inefficient (Sambrook, J.,Fritsch, E. F. & Maniatis, T. (1989) Molecular Cloning (Cold SpringHarbor Laboratory Press, Cold Spring Harbor)). They commonly involveseveral in vitro steps of DNA manipulation. Finding the desired endproduct requires arduous screening of this DNA after it has beentransformed into a suitable host, usually Escherichia coli.Nevertheless, many aspects of molecular genetics rely heavily upon suchmethods.

Take, for example, the generation and cloning of random but localizedmutations (Shortle, D., DiMaio, D. & Nathans, D. (1981) Annu. Rev.Genetics 15, 265-294). Such an undertaking usually begins withmutagenesis, either chemical or otherwise, of the target DNA sequence.The target might be one specific gene, which in E. coli would be 1 kb inlength, on average. Often, a plasmid-borne copy of this gene is thetarget of mutagenesis. Mutations generated in this way have already beencloned, but could include changes in the cloning vector as well as inthe gene of interest. Alternatively, the target to be mutagenized mightbe a piece of DNA containing this gene and nothing else. To be useful,mutations generated in this way must still be cloned and identified.

It is not always possible to rig a mutagenic treatment to generate thepreferred frequency of mutational events. Too low a mutation rateincreases the amount of work required to find changes of interest. Toohigh a mutation rate requires that one distinguish which of multiplebase changes are responsible for the mutant phenotype. In any event, theimportance of specific base changes must be verified. Traditionalmethods for doing this involve physically separating multiple changes,given the availability of useful restriction sites, or reconstructingeach mutant separately by one of any number of protocols forsite-directed mutagenesis (Herlitze, S. & Koenen, M. (1990) Gene 91,143-147; Kunkel, T. A. (1985) Proc. Natl. Acad. Sci. USA 82, 488-492.;Zoller, M. J. & Smith, M. (1987) Methods Enzymol. 154, 329-350.-5), allof which require at least one primer per specific mutation.

In a separate report, we will describe our use of the two-hybrid system(Chien, C.-t., Bartel, P. L., Sternglanz, R. & Fields, S. (1991) Proc.Natl. Acad. Sci. USA 88, 9578-9582.; Fields, S. & Song, O.-k. (1989)Nature (London) 340, 245-246) to identify a strong interaction betweentwo components of the flagellar motor of E. coli, FliG and FliM, and ourmutational analysis of this interaction (D. L. Marykwas and H. C. Berg,manuscript in preparation). Here we describe a facile method for mappingthe location of mutations within genes. We illustrate this method byapplication to interaction-defective mutations in fliG.

MATERIALS AND METHODS

Strains and Plasmids. S. cerevisiae strain GGY1::171 (Gill, G. &Ptashne, M. (1987) Cell 51, 121-126) was used for two-hybrid screening(Chien, C.-t., Bartel, P. L., Sternglanz, R. & Fields, S. (1991) Proc.Natl. Acad. Sci. USA 88, 9578-9582). E. coli strain DH5α (from BethesdaResearch Laboratories) was used for most traditional cloning. PlasmidpBD-G^(WT) encoded the GAL4 DNA-binding domain fusion to wild-type (WT)FliG. Plasmid pAD-M^(WT) encoded the GAL4 activation domain fusion to WTFliM. The construction of these plasmids will be described elsewhere (D.L. Marykwas and H. C. Berg, manuscript in preparation), as will be theisolation of fliG mutant derivatives of plasmid pBD-G^(WT), namedpBD-G¹⁰, pBD-G¹⁵, and pBD-G²⁵, to represent mutants 10, 15, and 25,respectively. Plasmid pMA424 is the GAL4 DNA-binding domain cloningvector described by Ma and Ptashne (Ma, J. & Ptashne, M. (1987) Cell 51,113-119).

DNA Amplification. PCR primers (Table 1) were supplied by Integrated DNATechnologies. Modified Pfu polymerase was from BioInsight. A typicalamplification reaction was performed in a total volume of 100 μlcontaining 0.02 to 2 ng of template plasmid DNA, 25 nmoles of each dNTP,20 pmoles of each primer, and 4 units of polymerase in the bufferrecommended by the enzyme manufacturer. The magnesium concentrationoften had to be optimized, the final concentration usually rangingbetween 2 and 10 mM. Reactions were performed in a MiniCycler from MJResearch as follows: 94° C. for 5 min; 25 cycles of 94° C. for 45 s, 50°C. for 1.5 min, 72° C. for 1.5 min; 72° C. for 5 min. Sometimes theannealing temperature was different than 50° C., determined either bythe melting temperature of the primers involved or empirically. Reactionproducts were examined for yield and purity by gel electrophoresis andcleaned up over QIAquick columns (from QIAGEN) prior to use. Removal ofreaction buffer, nucleotides and primers was not necessary for in vivomapping, but was required if the reaction products were to be sequenced.

Yeast Transformations. Yeast cells were grown on complete syntheticdropout medium (CSM-deleted component) containing per liter: 6.7 g yeastnitrogen base without amino acids, Difco; 1X drop-out supplement mixture(defining the deleted component), BIO101; and 20 g glucose. Yeast cellswere transformed following the single-stranded carrier method of Gietzand Schiestl (Gietz, R. D. & Schiestl, R. H. (1991) Yeast 7, 253-263).CSM-his lacks histidine, allowing the selection of His+ transformants.CSM-leu lacks leucine, allowing the selection of Leu+ transformants.CSM-his-leu lacks both histidine and leucine, allowing double selection.In a given mapping experiment, GGY1::171 yeast cells already harboringpAD-M^(WT) were cotransformed with 5 μl of each PCR product (10 to 75 ngeach) and 1 μl (33 to 100 ng) of linear gapped pMA424 (prepared byrestriction enzyme digestion with both EcoRI and BamHI, followed byphenol extraction and ethanol precipitation). The resident plasmidpAD-M^(WT) contained the selectable LEU2 gene. The recombinant plasmidconstructed in vivo, derived from pMA424, contained the HIS3 gene.Therefore, transformants containing both plasmids were selected at 30°C. on CSM-his-leu plates.

Two-hybrid Screening. Yeast transformants were scored for theirinteraction phenotype on SSX-his-leu plates (CSM-his-leu plates with0.1M KH₂PO₄, pH 7.0, 0.05% [wt/vol] 5-bromo-4-chloro-3-indolylβ-D-galactoside [X-gal], and sucrose in place of glucose). Thetransformants were directly lifted onto Millipore HA filters and placedcolony-side-up onto the SSX-his-leu plates, which were then incubated at30° C. Color was scored after 1 or 2 days. Usually whole plates weretested, each representing the in vivo recombination of one mutant genesegment with the remaining WT gene segment. The reciprocal experimentwas then tested on a separate SSX-his-leu plate. Sometimes side-by-sidecomparison of reciprocal experiments were performed on the same plate bylifting colonies onto smaller Millipore HA filters (25 instead of 85 mmdia.), several of which easily fit on a plate. However, fewerindependent recombinants could be sampled in this way.

Plasmid Rescue. Plasmid DNA was extracted from yeast cells by the methodof Ward (Ward, A. C. (1990) Nucleic Acids Res. 18, 5319). The plasmidsto be recovered that contained the subcloned fliG alleles all carriedthe HIS3 gene; therefore, cells containing these recombinant subcloneswere grown under His+ selection. One μl of the extracted DNA was thentransformed into E. coli strain DH5α, made electrocompetent as describedby Dower et al. (Dower, W. J., Miller, J. F. & Ragsdale, C. W. (1988)Nucleic Acids Res. 16, 6127-6145); ampicillin-resistant colonies wereselected. Although grown under His+ selection and not Leu+ selection,the yeast cells might also have harbored the LEU2-containing plasmidpAD-M^(WT). Plasmids isolated from E. coli transformed with eitherHIS3-containing plasmid DNA (the recombinant) or LEU2-containing plasmidDNA were easy to distinguish electrophoretically, because the plasmidswere of different size and had different restriction patterns.

DNA Sequence Analysis. Plasmid DNA was sequenced following thedouble-stranded plasmid sequencing protocol of Del Sal et al. (Del Sal,G., Manfioletti, G. & Schneider, C. (1989) Biotechniques 7, 514-519),except that we used Sequenase, [³³Pα]dATP, and as template {fraction(1/10)}th of the DNA obtained from a 1.5 ml boiling miniprep. PCRproducts were sequenced using the procedure described by Thein (Thein,S. L. (1989) Comments 16, 8), with [³³Pα]dATP.

RESULTS

The Method, in Principle. Our mapping method takes advantage of thehighly efficient recombination system found in S. cerevisiae (Muhlrad,D., Hunter, R. & Parker, R. (1992) Yeast 8, 79-82.; Rothstein, R. (1991)Methods Enzymol. 194, 281-301). We have found that yeast can repairplasmid gaps using not only one homologous fragment, but multiplefragments with overlapping homology. This feature has allowed us toconstruct hybrid genes in vivo, derived partly from the mutant andpartly from the WT, unrestricted by the availability of convenientrestriction sites.

The DNAs used for the development of this method express two componentsof the flagellar motor of E. coli that interact, FliG and FliM (D. L.Marykwas and H. C. Berg, manuscript in preparation). When FliG fused tothe DNA-binding domain of GAL4 and FliM fused to a transcriptionalactivation domain of GAL4 were coexpressed in yeast, they reconstituteda functional GAL4-like transcription factor that activated theexpression of a GAL4-dependent lacZ reporter gene. We also generated acollection of plasmid-borne FliG mutants (fused to the DNA-bindingdomain) that interact with FliM less well than does WT FliG (D. L.Marykwas and H. C. Berg, manuscript in preparation). To identify thefliG mutations responsible for the reduced FliG/FliM interaction, wedeveloped the method described here and illustrated in FIG. 3.

We have made chimeras of mutant and WT fliG genes (fused in frame withDNA encoding GAL4's DNA-binding domain). We generated the parts via PCRusing modified Pfu polymerase, which performs more faithful proofreadingthan does Taq polymerase. The parts were designed to overlap to providehomology. The non-overlapping ends have homology with the cloning vector(in this case the DNA-binding domain vector). All three pieces together,without ligase, are transformed into yeast and undergo in vivorecombination. The activation domain fusion to FliM, and the reportergene, are already present, allowing direct screening of the bluephenotype.

FIG. 3 shows that when both parts are derived from the WT fliG fusion,in vivo recombination with the binding domain vector results in cellsthat are dark blue, the WT phenotype, indicating a positive FliG/FliMinteraction. In the example illustrated, when the left part is derivedfrom WT and the right part is from the mutant, in vivo recombinationalso results in a WT dark blue phenotype. However, in the converseexperiment, when the left part is derived from the fliG mutant and theright part is from WT, the result is light blue, the mutant phenotype.Therefore, in the example shown, the fliG mutation responsible forreducing the FliG/FliM interaction maps to the left part of the mutantgene. If any fliG mutations are present in the right half of the fliGmutant, they are silent in this assay and therefore not responsible forthe reduced FliG/FliM interaction.

Strategy to Map fliG Mutants. We isolated 18 interaction-defective fliGmutants (D. L. Marykwas and H. C. Berg, manuscript in preparation). Ourstrategy to map these mutations is illustrated in FIG. 4. The fliGcoding region is 996 bp in length, including the stop codon. PrimeroGAL132-137 primes within GAL4 sequences upstream of the fliG codingsequence in our clones (pBD-G^(WT), pBD-G¹⁰, pBD-G¹⁵, and pBD-G²⁵).oADHterm primes within the ADH1 transcriptional terminator locateddownstream of the fliG coding sequence in our clones. Primers mapoGa,mapoGb, mapoGc, and mapoGd prime within the fliG coding sequence. Theseprimers were used to amplify various parts of fliG. PCR with primersoGAL132-137 and mapoGb gave fragment IB, containing the first third offliG. PCR with primers mapoGa and oADHterm gave fragment AT, containingthe remaining two-thirds of fliG. Primers mapoGa and mapoGb prime onopposite strands of fliG, providing fragments IB and AT with a region ofoverlap 107 bp in length. Similarly, fragment ID, containing the firsttwo-thirds of fliG, was generated with primers oGAL132-137 and mapoGd,whereas fragment CT, containing the remaining third of fliG, wasgenerated with primers mapoGc and oADHterm, with a region of overlap 99bp in length. The non-overlapping ends of each pair of overlappingfragments share homology (52 bp and 192 bp) with pMA424 (the vector forthe DNA-binding domain).

For each fliG mutant to be mapped, hybrids were generated by the in vivorecombination (with linear gapped pMA424) of mutant IB with WT AT, andcompared to the reciprocal recombination of mutant AT with WT IB.Another pair of reciprocal hybrids were generated combining WT andmutant ID and CT fragments. The information obtained from these fourcombinations was enough to determine whether the fliG change responsiblefor the interaction-defective mutant phenotype laid within the first,second, or third portion of the fliG gene. In effect, we have dividedthe fliG gene into three regions of similar size, the ends of which weredefined by the PCR primers used to generate the mapping fragments.

A Simple Case. Table 2 shows mapping data for three representative fliGmutants. One of these was mutant 25. The mutation responsible for theinteraction-defective phenotype of this mutant did not map to segmentIB²⁵ (IB amplified from pAD-G²⁵ as template), containing the first thirdof the mutant fliG gene, nor to CT²⁵, containing the last third of themutant fliG gene. Instead, the responsible change mapped to segmentsAT²⁵ and ID²⁵. The in vivo recombinants generated with either of thesemutant segments were light blue (the mutant phenotype) when tested forthe FliG/FliM two-hybrid interaction on X-gal plates. These two segmentshave in common the middle third of the mutant fliG gene. Indeed, DNAsequencing of mutant 25 has revealed a single base change in thisregion. It results in a his155→pro substitution (sequence data notshown).

A More Complicated Example. fliG mutant 15 clearly mapped to segmentAT¹⁵, not to segment IB¹⁵ (Table2). However, in vivo recombination usingsegments ID and CT led to a mixed population of both mutant and WTrecombinants, in about equal ratio, whether the mutant fragment was ID¹⁵or CT¹⁵. This mutation did not map clearly to either one of thesefragments. Instead, the mapping data suggest that fliG mutant 15 maps tothe region of overlap between fragments ID and CT. Indeed, DNAsequencing of mutant 15 has revealed a single base change in this regionspanning the 99 bp overlap. It results in a leu225→pro substitution(sequence data not shown).

Separating Two Closely Linked Base Changes. fliG mutant 10 also mappedto the middle third of the mutant gene (Table 2). However, DNAsequencing revealed two closely linked single base changes in the mutantgene (not shown), separated by only 35 bp. As each change resulted in anamino acid substitution (gln141→arg; leu153→pro), we were not able todetermine, a priori, which of the two changes was responsible for themutant phenotype. To distinguish the two possibilities, we generatedhybrids that contained one or the other but not both of these changes.The forward primer mapoGl and the reverse primer mapoGn recognize thesequence that separates the two base changes but prime on oppositestrands of the gene. PCR with primers oGAL132-137 and mapoGn gavefragment IN, whereas PCR with mapoGl and oADHterm gave fragment LT, withan overlap 33 bp in length. In vivo recombination using IN¹⁰ and LT^(WT)gave recombinants that appeared WT for the FliG/FliM interaction,whereas LT¹⁰/IN^(WT) recombinants exhibited the mutant phenotype(Table3). This determined that the L153P substitution was responsiblefor the interaction-defective phenotype of fliG mutant 10; the Q141Rsubstitution was phenotypically silent in this assay. As expected, thein vivo recombination of vector and mutant fragment LD¹⁰ with WTfragments IN^(WT) and CT^(WT) (4 pieces total) also yielded mutantrecombinants (Table 3). Plasmid DNA isolated from these cells wassequenced and found to contain the expected single base change.

DISCUSSION

We have described a new way to map mutations in DNA by multifragment invivo cloning. We have illustrated this method by mapping fliG mutationsthat disrupt the FliG/FliM two-hybrid interaction. Hybrids of mutant andWT fliG genes were created in yeast in vivo by homologous recombinationand scored directly for their interaction phenotypes. Since yeast cellsare transformed under conditions of no growth, each recombinant providedan independent test of the location of the mutation being mapped. Forexample, the 479 light blue ID²⁵/CT^(WT) recombinants (Table 2)represent 479 separate tests showing that mutation 25 mapped to segmentID. If traditional cloning methods had been used, comparable data couldhave been obtained only by constructing 479 independent subclones invitro, testing these 479 subclones in vivo by transforming themseparately into yeast, and then scoring their phenotypes.

Mutations can be mapped by multifragment in vivo cloning to a highdegree of resolution. We chose to determine if our fliG mutants mappedto the first, second, or last third of the 996 bp fliG gene and designedprimers accordingly. We felt that a 300 bp map segment was small enoughto sequence efficiently. However, one can divide a gene into as many mapsegments as desired, limited only by the number of primers used. One ofthe mapped mutations described (mutant 15) was localized to a small 99bp region. This was possible due to its fortuitous map location in thehomologous region of overlap between two mapping segments. Therefore,the smallest region to which a mutation can be mapped (in oneexperiment) is limited by the smallest amount of homology required foroverlapping map segments to recombine in vivo. We have not yet definedthe lowest limit of overlap necessary for recombination; however, a 33bp overlap is sufficient.

Occasionally, a mutation might be covered by one of the mapping primers.Indeed, this has been the case with some of our fliG mutants(unpublished data). These still map to the region of overlap between themap segments involved. However, we have found that modified Pfupolymerase does not always repair primer mismatches as little as 1 bpremoved from the annealed 3′ end. Therefore, if a WT primer covers amutation, that primer will usually be extended, yielding a PCR productthat is WT for the covered mutation.

An alternative method for recombinational mapping of plasmid-borne genesin yeast has already been described (Kunes, S., Ma, H., Overbye, K.,Fox, M. S. & Botstein, D. (1987) Genetics 115, 73-81). It involvestesting the ability of an individual restriction fragment to correct aplasmid-borne mutation by gene conversion. Our method of multifragmentin vivo cloning differs from that of Kunes et al. in many ways. We usemultiple overlapping fragments, not just one fragment, to close aplasmid gap in vivo. In this way, we construct plasmid subclones thatacquire the mutation of interest from a specific DNA fragment, withsuccess rates of nearly 100%. By contrast, the method of Kunes et al.relies upon the incidence of loss of a plasmid-borne mutation toidentify its location; in the end, the mutation is lost, not subcloned.Our method, the construction and testing of reciprocal hybrids in vivo,provides redundant information that should identify both where theresponsible change is and where it is not. Kunes' method fails to do so.This is another important difference. A mutant gene might containmultiple base changes; each could have a mutant phenotype or they couldact together to create one. This information can be reconstructed usingour method but would be lost with theirs. Alternatively, all but onemutation might be silent and located in regions sequenced outside of theidentified map location. Such silent mutations might reveal the extentto which certain parts of a gene may be altered without affecting thespecific gene function being assayed. Indeed, we have found several fliGmutations that do not significantly affect the FliG/FliM two-hybridinteraction (D. L. Marykwas and H. C. Berg, manuscript in preparation).Identification of such regions should prove informative, but would nothave been possible had we used the method of Kunes et al.

Multifragment in vivo cloning relies heavily upon the use of PCR andtherefore shares many of the same pitfalls. Since our primary use is formapping mutations, we reduce the likelihood of creating additionalmutations by using a thermophilic enzyme that does not introduce errorstoo frequently. Based on our cumulative sequencing of cloned PCRproducts, we have found <1 base change for every 10 kb of modifiedPfu-amplified DNA, compared to 1 base change for every 280 bp amplifiedby Taq polymerase under the conditions we use. Nevertheless, althoughmodified Pfu polymerase proofreads, errors are still made and sometimesvisible in our mapping data. Mapping by multifragment in vivo cloning issensitive to the presence of starting template in the PCR-derivedmapping fragments. Our starting templates are plasmids encoding mutantor WT FliG fused to the GAL4 DNA-binding domain. Any template moleculestransformed into yeast will provide a background of transformants thatexhibit the template-dependent phenotype. To avoid excessivetemplate-related background problems, we use very little startingtemplate, or we perform two consecutive amplifications, using thereaction products of the first amplification as the template for thesecond. Obviously, multifragment in vivo cloning could also giveambiguous results if the template was a mixed population of mutant andWT plasmids. Finally, not every pair of overlapping fragments givesequivalent transformation frequencies, we believe when either fragmentis limiting due to poor amplification via some primer pairs.

There are some problems associated with multifragment in vivo cloningthat are unrelated to PCR. We always have a background of tan yeasttransformants that do not display reporter gene activity on indicatorplates (see Tables 2 and 3). Based on control transformations, weattribute these to incompletely cut vector and to illegitimaterecombination of the vector; we have observed both. We have also seenvariable efficiencies of in vivo recombination, even when the same DNAwas used, but on different days with different cell cultures. Thisvariability appeared to be due to differences in yeast cell competence,as transformation with intact plasmid appeared to change in the sameway.

There have been several recent reports of in vivo cloning in E. coli(Jones, D. H. & Howard, B. H. (1990) Biotechniques 8, 178-183; Jones, D.H. & Howard, B. H. (1991) Biotechniques 10, 62-66; Jones, D. H. &Winistorfer, S. C. (1992) Biotechniques 12, 528-535; Oliner, J. D.,Kinzler, K. W. & Vogelstein, B. (1993) Nucleic Acids Res). None of thesemethods have been applied to the mapping of mutations. Nor have anyinvolved the recombination of more than two DNA fragments. Our mappingis routinely done with three pieces (the vector plus two overlappinginserts) and has also worked quite well with four (see Table 3). Therecent reports all cite a requirement for absolute homology at the freeends of the vector being recombined. Although homology is also requiredfor our in vivo recombination of multiple fragments in yeast, thehomology is not strictly necessary at either end of our gapped vector orinsert and may be internal at each end. Ends with as few as 2 and asmany as 327 non-homologous base pairs work just as well as ends withabsolute homology (our unpublished observations).

Finally, our multifragment in vivo cloning is much more efficient thanany of the published procedures, even those described to work in Rec+strains of E. coli. We transformed yeast with linear vector DNA and afull length fliG-containing piece with homology to the cloning vector atboth of its ends. Using the same DNA, we transformed two differentrecombination-proficient strains of E. coli, including a recD strainreported to allow efficient uptake and recombination of linear DNA. Wealso compared the transformation of yeast versus E. coli with threeoverlapping pieces, as in most of our mapping experiments. We wereunable to detect recombination products of three DNA fragments in E.coli. We obtained a single recombinant of two pieces from E. coli, butmany thousands from the same DNA transformed into S. cerevisiae (datanot shown).

In conclusion, multifragment in vivo cloning provides an easy way to mapmutations. It facilitates the physical separation of multiple changes ina target sequence. The converse should also be true. It should simplifythe joining of multiple base changes into one composite multi-mutant, asmight be required to perform accelerated in vivo evolution experiments.As we will report in a separate article, the same principles we use tomap and subclone mutations can be applied to constructing complexcustom-made plasmids by the in vivo recombination of DNA cassettesdesigned to overlap. Therefore, multifragment in vivo cloning provides anew approach towards genetic manipulation that should eliminate stepsrequiring bacterial hosts and the foreign DNA sequence elementsnecessary for replication and selection in these hosts. Thus, beer andwine makers and other workers in the agricultural industry mightgenetically engineer a better product in a manner that is commerciallyacceptable. Multifragment in vivo cloning is a powerful mapping tool,especially when combined with the two-hybrid system. However, it shouldbe possible to use this method in any transformable organism whereefficient homologous recombination is possible and where mutants andnon-mutants are distinguishable.

FIG. 3. Mapping mutations using in vivo recombination. The WT gene isrepresented by open rectangles, the mutant gene by the filledrectangles. The mutation to be mapped is indicated by an asterisk. PCRprimers are shown as arrows. One pair of primers is used to amplify theleft part of each gene. A second pair of primers is used to amplify theright part. The gene parts overlap with each other and with a cloningvector, shown by the ovals. The regions of overlap allow homologousrecombination as indicated by an X. Hybrids are created in vivo by therecombination of mutant and WT fragments, and then scored directly fortheir phenotype. Pairs of reciprocal hybrids are tested, for comparison.Both WT fragments are also recombined, as a positive control. In theexample shown, the mutation is in the left fragment of the mutant gene.

FIG. 4. Strategy used to map fliG mutants. The top bar represents thegene organization of fliG in pBD-G^(WT). The next bar represents a fliGmutant derived from this plasmid. GAL4 bd encodes the GAL4 DNA-bindingdomain. ADH1 term is a transcription termination sequence from the yeastADH1 gene. The arrows represent the relative positions of PCR primers(described in Table 1) used to amplify various parts offliG:I=oGAL132-137, T=oADHterm, A=mapoGa, B=mapoGb, C=mapoGc, andD=mapoGd. Regions of overlap that allow homologous recombination in vivoare each indicated by an X. The pairs of gene fragments that arerecombined in vivo are shown. Each fragment is defined (and labeled) bythe primers used for its amplification. Hybrids of mutant and WT fliGare created by recombining one mutant fragment with one WT fragment.Pairs of reciprocal hybrids are always tested, for comparison.

TABLE 1 Primers used in this study Name Sequence Priming siteoGAL132-137 5′-tcatcggaagagagtagt-3′ 394 → 411 oADHterm5′-gagcgacctcatgctatacc-3′ 1213 → 1194 mapoGa 5′-agatattctcgaaactcg-3′285 → 302 mapoGb 5′-cgataatttgcggatgct-3′ 391 → 374 mapoGc5′-ctgatgaaaactcagcag-3′ 613 → 630 mapoGd 5′-ctcgaacaggaacatctc-3′ 711 →694 mapoGl 5′-gccgccgatattctggcgt-3′ 424 → 442 mapoGn5′-acgttcatcgaacaac-3′ 456 → 441

The priming site relative to +1 of the respective structural gene: GAL4for oGAL132-137, ADH1 for oADHterm, and fliG for mapoGa-mapoGn.

TABLE 2 Representative mapping data Mutant fliG mutant data fragment 1015 25 IB  30 DB (100%) 111 DB (97.4%)  34 DB (100%)  15 tan  3 LB (2.6%) 26 tan 100 tan AT  15 LB (100%)  9 LB (100%)  18 LB (100%)  16 tan  77tan  30 tan ID 225 tan 184 DB (51%)  8 M/DB (1.6%) 175 LB (48.5%) 479 LB(98.4%)  2 MB (0.5%) 122 tan  67 tan CT 263 DB (98.5%) 200 DB (51.5%)107 DB (97.3%)  4 LB (1.5%) 188 LB (48.5%)  3 MB (2.7%)  96 tan 112 tan111 tan

fliG mutants 10, 15, and 25 were mapped by in vivo recombination asdescribed in the text. Mutant IB fragments were recombined with AT^(WT).Mutant AT fragments were recombined with IB^(WT). Mutant ID fragmentswere recombined with CT^(WT). Mutant CT fragments were recombined withID^(WT). For each pair of mutant and WT fragments so joined, the totalnumber of transformants displaying each phenotype is given, followed inparentheses by that same number expressed as the percentage of totalrecombinants. LB is light blue. MB is medium blue. DB is dark blue. Tantransformants are not considered to be recombinants (see Discussion).The in vivo recombination of ID¹⁰ with CT^(WT) never gave anything buttan transformants. More definitive fliG mutant 10 mapping data is shownin Table 3.

TABLE 3 More fliG mutant 10 mapping data Fragments combined in vivoRecombinant phenotypes IN^(WT) + LT¹⁰  0 DB  23 LB (100%) 115 tan IN¹⁰ +LT^(WT)  68 DB (97%)  2 LB (3%)  96 tan IN^(WT) + LD^(WT) + CT^(WT) 103DB (100%)  0 LB 500 tan IN¹⁰ + LD¹⁰ + CT¹⁰  7 DB (2.6%) 260 LB (97.4%)550 tan IN¹⁰ + LD^(WT) + CT^(WT) 700 DB (99.4%)  4 M/LB (0.6%) 520 tanIN^(WT) + LD¹⁰ + CT^(WT)  2 DB (5%)  38 LB (95%) 560 tan IN^(WT) +LD^(WT) + CT¹⁰ 241 DB (97.6%)  6 LB (2.4%) 506 tan

Mapping fragments were amplified from either WT or mutant 10 templateDNA. Each mapping fragment is named after the primers and template usedfor its amplification. For example, IN^(WT) fragments were amplifiedfrom WT template using primers oGAL132-137 (I) and mapoGn (N). Likewise,LT¹⁰ fragments were amplified from mutant 10 template using primersmapoGl (L) and oADHterm (T). Other primers used were mapoGc (C) andmapoGd (D).

Conclusion, Ramifications, and Scope

In particular, the present invention relates to a general method forcloning that can be practiced using any organism with a suitablyefficient and precise in vivo recombination system.

This method can be used to construct plasmids that are composed entirelyof DNA isolated from the host organism, provided suitable DNAreplication and selectable markers are available for use in thatorganism.

This method can serve as the basis for a cassette-based cloning system,where suitably designed plasmids serve as sources for 1) linearfragments containing DNA replication elements flanked by specific DNAsequence elements that serve as recombination tags ,and 2) linearfragments containing selectable markers flanked by specific DNA sequenceelements that serve as recombination tags. Thus plasmid constructionscan be greatly simplified by the use of such a cassette-based cloningsystem.

The present invention also relates to a method of mapping mutations.This is both a rapid and simple method for mapping mutations. We haveused this method to map the position of mutations that disrupted atwo-hybrid interaction between the E. coli proteins FliG and FliM.

Additionally, the invention relates to a method of recombination thatcan be used to reassort mutations in vivo and thus can facilitatedirected-evolution by multiple iterations of a procedure that selectsfor and then reassorts mutations.

Although the descriptions above contain many specificities, these shouldnot be construed as limiting the scope of the invention but as merelyproviding some of the presently preferred embodiments of this invention.

Thus, the scope of the invention should be determined by the appendedclaims and their legal equivalents, rather than by the examples given.

8 18 base pairs nucleic acid single linear Other nucleic acid (SyntheticDNA) unknown 1 TCATCGGAAG AGAGTAGT 18 20 base pairs nucleic acid singlelinear Other nucleic acid (Synthetic DNA) unknown 2 GACCGACCTCATGCTATACC 20 18 base pairs nucleic acid single linear Other nucleicacid (Synthetic DNA) unknown 3 AGATATTCTC GAAACTCG 18 18 base pairsnucleic acid single linear Other nucleic acid (Synthetic DNA) unknown 4CGATAATTTG CGGATGCT 18 18 base pairs nucleic acid single linear Othernucleic acid (Synthetic DNA) unknown 5 CTGATGAAAA CTCAGCAG 18 18 basepairs nucleic acid single linear Other nucleic acid (Synthetic DNA)unknown 6 CTCGAACAGG AACATCTC 18 19 base pairs nucleic acid singlelinear Other nucleic acid (Synthetic DNA) unknown 7 GCCGCCGATA TTCTGGCGT19 16 base pairs nucleic acid single linear Other nucleic acid(Synthetic DNA) unknown 8 ACGTTCATCG AACAAC 16

What is claimed is:
 1. A method of directed in vivo DNA recombination,resulting in the production of a circular double-stranded DNA moleculecomprising the steps of: (i) in a first container means containing afirst aliquot of a full-length mutant 1 double-stranded DNA amplifying afirst double-stranded mutant 1 DNA segment by means of the PCR process,wherein a plurality of primers are provided so that two primers effectsaid amplification; (ii) in a second container means a linear gappedacceptor vector is generated by linearizing a vector, capable ofreplication and selection in a host with an efficient and precise invivo homologous recombination system, using appropriate restrictionenzyme(s); (iii) in a first container means containing a first aliquotof a full-length mutant 2 double-stranded DNA amplifying a firstdouble-stranded mutant 2 DNA segment by means of the PCR process,wherein a plurality of primers are provided so that two primers effectsaid amplification, and wherein said first double-stranded mutant 1 DNAsegment and said first double-stranded mutant 2 DNA segment arehomologous within the region to be recombined, and wherein one end ofsaid first double-stranded mutant 1 DNA segment is homologous to an endof a linear gapped acceptor plasmid, and wherein one end of said firstdouble-stranded mutant 2 DNA segment is homologous to the other end ofsaid linear gapped acceptor vector; (iv) transforming the product ofstep (i), and the product of step (ii), and the product of step (iii)together into a host with a suitable, efficient, and accurate in vivorecombination system, and allowing said products to recombine in vivo atthe homologous sequences, thereby producing a collection of recombineddouble-stranded circular DNA molecules.
 2. The method of claim 1wherein: (i) the recombination process is carried out initially using apool of mutated DNA, encoding an activity to be evolved, as thesubstrate for the PCR amplification; (ii) the products of the in vivoDNA recombination process are incorporated into a vector compatible witha host strain used to identify the improved activity; (iii) a multitudeof independently derived products of the in vivo DNA recombinationprocess are examined in the host strain, and isolates bearing vectorsencoding the improved activity are identified and isolated; (iv) therecombination process is carried out iteratively, the mutation-bearingDNAs identified in step (iii) are pooled and used as the pool of mutatedDNA of step (i), allowing for an incremental, combinatorial search formultiple mutations that improve function.
 3. The method of claim 1wherein: (i) the host organism is yeast.
 4. The method of claim 1wherein: (i) the linear gapped acceptor vector of step (ii) is generatedby the PCR process, wherein a plurality of primers are provided so thattwo primers effect said amplification.
 5. The method of claim 1 whereinthe third step is: (iii) in a first container means containing a firstaliquot of a full-length mutant 2 double-stranded DNA amplifying a firstdouble-stranded mutant 2 DNA segment by means of the PCR process,wherein a plurality of primers are provided so that two primers effectsaid amplification, and wherein said first double-stranded mutant 1 DNAsegment and said first double-stranded mutant 2 DNA segment arehomologous with one or more additional dsDNA segment(s) within theregions to be recombined, and wherein one end of said firstdouble-stranded mutant 1 DNA segment is homologous to an end of a lineargapped acceptor plasmid, and wherein one end of said firstdouble-stranded mutant 2 DNA segment is homologous to the other end ofsaid linear gapped acceptor vector.
 6. The method of claim 5 wherein thehost organism is yeast.
 7. The method of claim 5 wherein additionaldouble-stranded mutant DNA segment(s) are amplified by means of the PCRprocess, wherein a plurality of primers are provided so that two primerseffect said amplification.
 8. The method of claim 5 wherein saidadditional double-stranded mutant DNA segment(s) are derived by cleavagewith restriction enzyme(s).
 9. The method of claim 2 wherein at leastone additional mutant dsDNA segment with homology to one or more othersegments is transformed into a host with a suitable, efficient, andaccurate in vivo recombination system.
 10. The method of claim 9 whereinthe host organism is yeast.